Epidemiological characteristics of seven notifiable respiratory infectious diseases in the mainland of China: an analysis of national surveillance data from 2017 to 2021.

BACKGROUND: Respiratory infectious diseases (RIDs) remain a pressing public health concern, posing a significant threat to the well-being and lives of individuals. This study delves into the incidence of seven primary RIDs during the period 2017-2021, aiming to gain deeper insights into their epidemiological characteristics for the purpose of enhancing control and prevention strategies. METHODS: Data pertaining to seven notifiable RIDs, namely, seasonal influenza, pulmonary tuberculosis (PTB), mumps, scarlet fever, pertussis, rubella and measles, in the mainland of China between 2017 and 2021 were obtained from the National Notifiable Disease Reporting System (NNDRS). Joinpoint regression software was utilized to analyze temporal trends, while SaTScan software with a Poisson probability model was used to assess seasonal and spatial patterns. RESULTS: A total of 11,963,886 cases of the seven RIDs were reported during 2017-2021, and yielding a five-year average incidence rate of 170.73 per 100,000 individuals. Among these RIDs, seasonal influenza exhibited the highest average incidence rate (94.14 per 100,000), followed by PTB (55.52 per 100,000), mumps (15.16 per 100,000), scarlet fever (4.02 per 100,000), pertussis (1.10 per 100,000), rubella (0.59 per 100,000), and measles (0.21 per 100,000). Males experienced higher incidence rates across all seven RIDs. PTB incidence was notably elevated among farmers and individuals aged over 65, whereas the other RIDs primarily affected children and students under 15 years of age. The incidences of PTB and measles exhibited a declining trend from 2017 to 2021 (APC = -7.53%, P = 0.009; APC = -40.87%, P = 0.02), while the other five RIDs peaked in 2019. Concerning seasonal and spatial distribution, the seven RIDs displayed distinct characteristics, with variations observed for the same RIDs across different regions. The proportion of laboratory-confirmed cases fluctuated among the seven RIDs from 2017 to 2021, with measles and rubella exhibiting higher proportions and mumps and scarlet fever showing lower proportions. CONCLUSIONS: The incidence of PTB and measles demonstrated a decrease in the mainland of China between 2017 and 2021, while the remaining five RIDs reached a peak in 2019. Overall, RIDs continue to pose a significant public health challenge. Urgent action is required to bolster capacity-building efforts and enhance control and prevention strategies for RIDs, taking into account regional disparities and epidemiological nuances. With the rapid advancement of high-tech solutions, the development and effective implementation of a digital/intelligent RIDs control and prevention system are imperative to facilitate precise surveillance, early warnings, and swift responses.

Metagenomic Analysis of Environmental Samples from Wildlife Rescue Station at Poyang Lake, China.

Objective: To improve the understanding of the virome and bacterial microbiome in the wildlife rescue station of Poyang Lake, China. Methods: Ten smear samples were collected in March 2019. Metagenomic sequencing was performed to delineate bacterial and viral diversity. Taxonomic analysis was performed using the Kraken2 and Bracken methods. A maximum-likelihood tree was constructed based on the RNA-dependent RNA polymerase (RdRp) region of picornavirus. Results: We identified 363 bacterial and 6 viral families. A significant difference in microbial and viral abundance was found between samples S01-S09 and S10. In S01-S09, members of Flavobacteriia and Gammaproteobacteria were the most prevalent, while in S10, the most prevalent bacteria class was Actinomycetia. Among S01-S09, members of Myoviridae and Herelleviridae were the most prevalent, while the dominant virus family of S10 was Picornaviridae. The full genome of the pigeon mesivirus-like virus (NC-BM-233) was recovered from S10 and contained an open reading frame of 8,124 nt. It showed the best hit to the pigeon mesivirus 2 polyprotein, with 84.10% amino acid identity. Phylogenetic analysis showed that RdRp clustered into Megrivirus B. Conclusion: This study provides an initial assessment of the bacteria and viruses in the cage-smeared samples, broadens our knowledge of viral and bacterial diversity, and is a way to discover potential pathogens in wild birds.

Epidemiological and virological surveillance of influenza viruses in China during 2020-2021.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, seasonal influenza activity declined globally and remained below previous seasonal levels, but intensified in China since 2021. Preventive measures to COVID-19 accompanied by different epidemic characteristics of influenza in different regions of the world. To better respond to influenza outbreaks under the COVID-19 pandemic, we analyzed the epidemiology, antigenic and genetic characteristics, and antiviral susceptibility of influenza viruses in the mainland of China during 2020-2021. METHODS: Respiratory specimens from influenza like illness cases were collected by sentinel hospitals and sent to network laboratories in Chinese National Influenza Surveillance Network. Antigenic mutation analysis of influenza virus isolates was performed by hemagglutination inhibition assay. Next-generation sequencing was used for genetic analyses. We also conducted molecular characterization and phylogenetic analysis of circulating influenza viruses. Viruses were tested for resistance to antiviral medications using phenotypic and/or sequence-based methods. RESULTS: In the mainland of China, influenza activity recovered in 2021 compared with that in 2020 and intensified during the traditional influenza winter season, but it did not exceed the peak in previous years. Almost all viruses isolated during the study period were of the B/Victoria lineage and were characterized by genetic diversity, with the subgroup 1A.3a.2 viruses currently predominated. 37.8% viruses tested were antigenically similar to reference viruses representing the components of the vaccine for the 2020-2021 and 2021-2022 Northern Hemisphere influenza seasons. In addition, China has a unique subgroup of 1A.3a.1 viruses. All viruses tested were sensitive to neuraminidase inhibitors and endonuclease inhibitors, except two B/Victoria lineage viruses identified to have reduced sensitivity to neuraminidase inhibitors. CONCLUSIONS: Influenza activity increased in the mainland of China in 2021, and caused flu season in the winter of 2021-2022. Although the diversity of influenza (sub)type decreases, B/Victoria lineage viruses show increased genetic and antigenic diversity. The world needs to be fully prepared for the co-epidemic of influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus globally.

Epidemiological characteristics of imported respiratory infectious diseases in China, 2014‒2018.

BACKGROUND: With the progress of globalization, international mobility increases, greatly facilitating cross-border transmission of respiratory infectious diseases (RIDs). This study aimed to analyze the epidemiological characteristics and factors influencing imported RIDs, with the goal of providing evidence to support adoption of high-tech, intelligent methods to early find imported RIDs and prevent their spread in China. METHODS: We obtained data of imported RIDs cases from 2014 to 2018 from the Inbound Sentinel Network of Customs and the National Notifiable Diseases Reporting System in China. We analyzed spatial, temporal, and population distribution characteristics of the imported RIDs. We developed an index to describe seasonality. Pearson correlation coefficients were used to examine associations between independent variables and imported cases. Data analyses and visualizations were conducted with R software. RESULTS: From a total of 1 409 265 253 inbound travelers, 31 732 (2.25/100 000) imported RIDs cases were reported. RIDs cases were imported from 142 countries and five continents. The incidence of imported RIDs was nearly 5 times higher in 2018 (2.81/100 000) than in 2014 (0.58/100 000). Among foreigners, incidence rates were higher among males (5.32/100 000), 0-14-year-olds (15.15/100 000), and cases originating in Oceania (11.10/100 000). The vast majority (90.3%) of imported RIDs were influenza, with seasonality consistent with annual seasonality of influenza. The spatial distribution of imported RIDs was different between Chinese citizens and foreigners. Increases in inbound travel volume and the number of influenza cases in source countries were associated with the number of imported RIDs. CONCLUSIONS: Our study documented importation of RIDs into China from 142 countries. Inbound travel poses a significant risks bringing important RIDs to China. It is urgent to strengthen surveillance at customs of inbound travelers and establish an intelligent surveillance and early warning system to prevent importation of RIDs to China for preventing further spread within China.

Distribution of avian influenza viruses according to environmental surveillance during 2014-2018, China.

BACKGROUND: Recurrent infections of animal hosts with avian influenza viruses (AIVs) have posted a persistent threat. It is very important to understand the avian influenza virus distribution and characteristics in environment associated with poultry and wild bird. The aim of this study was to analyze the geographic and seasonal distributions of AIVs in the 31 provinces, municipalities and autonomous region (PMA) of China, compare the AIVs prevalence in different collecting sites and sampling types, analyze the diversity of AIVs subtypes in environment. METHODS: A total of 742 005 environmental samples were collected from environmental samples related to poultry and wild birds in different locations in the mainland of China during 2014-2018. Viral RNA was extracted from the environmental samples. Real-time RT-PCR assays for influenza A, H5, H7 and H9 subtypes were performed on all the samples to identify subtypes of influenza virus. The nucleic acid of influenza A-positive samples were inoculated into embryonated chicken eggs for virus isolation. Whole-genome sequencing was then performed on Illumina platform. SPSS software was used to paired t test for the statistical analysis. ArcGIS was used for drawing map. Graphpad Prism was used to make graph. RESULTS: The nucleic acid positivity rate of influenza A, H5, H7 and H9 subtypes displayed the different characteristics of geographic distribution. The nucleic acid positivity rates of influenza A were particularly high (25.96%-45.51%) in eleven provinces covered the Central, Eastern, Southern, Southwest and Northwest of China. The nucleic acid positivity rates of H5 were significantly high (11.42%-13.79%) in two provinces and one municipality covered the Southwest and Central of China. The nucleic acid positivity rates of H7 were up to 4% in five provinces covered the Eastern and Central of China. The nucleic acid positivity rates of H9 were higher (13.07%-2.07%) in eleven PMA covered the Southern, Eastern, Central, Southwest and Northwest of China. The nucleic acid positivity rate of influenza A, H5, H7 and H9 showed the same seasonality. The highest nucleic acid positivity rates of influenza A, H5, H7, H9 subtypes were detected in December and January and lowest from May to September. Significant higher nucleic acid positivity rate of influenza A, H5, H7 and H9 were detected in samples collected from live poultry markets (LPM) (30.42%, 5.59%, 4.26%, 17.78%) and poultry slaughterhouses (22.96%, 4.2%, 2.08%, 12.63%). Environmental samples that were collected from sewage and chopping boards had significantly higher nucleic acid positivity rates for influenza A (36.58% and 33.1%), H5 (10.22% and 7.29%), H7(4.24% and 5.69%)and H9(21.62% and 18.75%). Multiple subtypes of AIVs including nine hemagglutinin (HA) and seven neuraminidase (NA) subtypes were isolated form the environmental samples. The H5, H7, and H9 subtypes accounted for the majority of AIVs in environment. CONCLUSIONS: In this study, we found the avian influenza viruses characteristics of geographic distribution, seasonality, location, samples types, proved that multiple subtypes of AIVs continuously coexisted in the environment associated with poultry and wild bird, highlighted the need for environmental surveillance in China.

Development and Assessment of Two Highly Pathogenic Avian Influenza (HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness.

OBJECTIVE: In China, 24 cases of human infection with highly pathogenic avian influenza (HPAI) H5N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5N6 candidate vaccine viruses (CVVs). METHODS: In accordance with the World Health Organization (WHO) recommendations, we constructed two reassortant viruses using reverse genetics (RG) technology to match the two different epidemic H5N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo. RESULTS: The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs. CONCLUSION: We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics, which may aid in our preparedness for a potential H5N6 pandemic.

Profile and generation of reduced neuraminidase inhibitor susceptibility in highly pathogenic avian influenza H7N9 virus from human cases in Mainland of China, 2016-2019.

Human infections with highly pathogenic avian influenza (HPAI) H7N9 virus were detected in late 2016. We examined the drug resistance profile of 30 HPAI H7N9 isolates from Mainland of China (2016-2019). Altogether, 23% (7/30) carried neuraminidase inhibitors (NAIs) - resistance mutations, and 13% (4/30) displayed reduced susceptibility to NAIs in neuraminidase (NA) inhibition test. An HPAI H7N9 reassortment virus we prepared was passaged with NAIs for 10 passages. Passage with zanamivir induced an E119G substitution in NA, whereas passage with oseltamivir induced R292K and E119V substitutions that simulated that seen in oseltamivir -treated HPAI H7N9 cases, indicating that the high frequency of resistant strains in the HPAI H7N9 isolates is related to NAIs use. In presence of NAIs, R238I, A146E, G151E and G234T substitutions were found in HA1 region of HA. No amino acid mutations were found in the internal genes of the recombinant virus.

Interferon-induced Transmembrane Protein 3 Prevents Acute Influenza Pathogenesis in Mice.

OBJECTIVE: Interferon-induced transmembrane protein 3 (IFITM3) is an important member of the IFITM family. However, the molecular mechanisms underlying its antiviral action have not been completely elucidated. Recent studies on IFITM3, particularly those focused on innate antiviral defense mechanisms, have shown that IFITM3 affects the body's adaptive immune response. The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection in vivo. METHODS: We performed proteomics, flow cytometry, and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer (NK) cell numbers, their activation, and their immune function in the lungs of Ifitm3-/- and wild-type mice. RESULTS: Ifitm3-/- mice developed more severe inflammation and apoptotic responses compared to wild-type mice. Moreover, the NK cell activation was higher in the lungs of Ifitm3-/- mice during acute influenza infection. CONCLUSIONS: Based on our results, we speculate that the NK cells are more readily activated in the absence of IFITM3, increasing mortality in Ifitm3-/- mice.

Serum tau levels are increased in patients with hyperthyroidism.

Hyperthyroidism may cause cognitive decline and increases the risk of Alzheimer's disease (AD), the major form of dementia; however, the underlying mechanism of this relationship is unclear. AD is associated with increased serum levels of tau. In this study, we investigated the relationship between serum thyroid hormones (THs) and tau. Fifty participants diagnosed with hyperthyroidism and fifty euthyroid counterparts were included and received clinical examinations. Serum concentrations of thyroid-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3) and tau protein were assessed. The total tau protein level was significantly higher in hyperthyroidism participants than in their euthyroid counterparts. The level of circulating total tau had a significant positive association with the serum concentrations of FT3 and FT4. Total tau level was increased in the low TSH group and the serum THs decreased with the increase of age. These findings reveal that peripheral THs are associated with the serum concentration of tau, which may be involved in the pathogenesis of AD, suggesting a potential therapeutic target of AD via hyperthyroidism therapy.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A (H5N1) Virus.

OBJECTIVE: To recover broad-neutralizing monoclonal antibodies (BnAbs) from avian influenza A (H5N1) virus infection cases and investigate their genetic and functional features. METHODS: We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients. The genetic basis, biological functions, and epitopes of the obtained BnAbs were assessed and modeled. RESULTS: Two BnAbs, 2-12D5, and 3-37G7.1, were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset. Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity (ADCC) activity. Albeit derived from distinct Ab lineages, i.e., V H1-69-D2-15-J H4 (2-12D5) and V H1-2-D3-9-J H5 (3-32G7.1), the BnAbs were directed toward CR6261-like epitopes in the HA stem, and HA 2 I45 in the hydrophobic pocket was the critical residue for their binding. Signature motifs for binding with the HA stem, namely, IFY in V H1-69-encoded Abs and LXYFXW in D3-9-encoded Abs, were also observed in 2-12D5 and 3-32G7.1, respectively. CONCLUSIONS: Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus. The HA stem epitopes targeted by the BnAbs, and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Effectiveness of favipiravir (T-705) against wild-type and oseltamivir-resistant influenza B virus in mice.

The emergence of resistant mutants to the wildly used neuraminidase inhibitors (NAIs) makes the development of novel drugs necessary. Favipiravir (T-705) is one of the RNA-dependent RNA polymerase (RdRp) inhibitors developed in recent years. To examine the efficacy of T-705 against influenza B virus infections in vivo, C57BL/6 mice infected with wild-type or oseltamivir-resistant influenza B/Memphis/20/96 viruses were treated with T-705. Starting 2 h post inoculation (hpi), T-705 was orally administered to mice BID at dosages of 50, 150, or 300 mg/kg/day for 5 days. Oseltamivir was used as control. Here, we showed that T-705 protected mice from lethal infection in a dose-dependent manner. T-705 administration also significantly reduced viral loads and suppressed pulmonary pathology. In addition, phenotypic assays demonstrated that no T-705-resistant viruses emerged after T-705 treatment. In conclusion, T-705 can be effective to protect mice from lethal infection with both wild-type and oseltamivir-resistant influenza B viruses.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine.

OBJECTIVE: To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine. METHODS: Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 µg/mouse) alone, or CTA1-DD (5 µg/mouse) alone, or H3N2 split vaccine (3 µg/mouse) plus CTA1-DD (5 µg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 µg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured. RESULTS: H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus. CONCLUSION: CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

Detection of reassortant avian influenza A (H11N9) virus in environmental samples from live poultry markets in China.

BACKGROUND: Avian influenza viruses have caused human infection and posed the pandemic potential. Live poultry markets are considered as a source of human infection with avian influenza viruses. Avian influenza routine surveillance of live poultry markets is taken annually in China. We isolated the 2 H11N9 influenza virus from the surveillance program. To better understand the risk caused by these new viruses, we characterize the genetic and pathogenicity of the two viruses. METHODS: Viral isolation was conducted with specific pathogen-free (SPF) embryonated chicken eggs. Whole genome was sequenced, and phylogenetic analysis was conducted. RESULTS: Two H11N9 viruses were identified, with all 8 segments belonging to the Eurasian lineage. The HA, NA, M, NS and PA genes were similar to virus isolates from ducks, and the NP, PB2 and PB1 gene segments were most similar to those viruses from wild birds, indicating that the H11N9 viruses might represent reassortant viruses from poultry and wild birds. The HA receptor binding preference was avian-like, and the cleavage site sequence of HA showed low pathogenic. The NA gene showed 94.6 % identity with the novel H7N9 virus that emerged in 2013. There was no drug resistance mutation in the M2 protein. The Asn30Asp and Thr215Ala substitutions in the M1 protein implied a potentially increased pathogenicity in mice. Both viruses were low-pathogenic strains, as assessed by the standards of intravenous pathogenicity index (IVPI) tests. CONCLUSION: Two reassortant H11N9 avian influenza viruses were detected. These viruses showed low pathogenicity to chickens in the IVPI test. Public health concern caused by the reassortant H11N9 viruses should be emphasized during the future surveillance.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing.

OBJECTIVE: To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform. METHODS: Virus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. RESULTS: The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus. CONCLUSION: The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China.

OBJECTIVE: To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China. METHODS: Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses. RESULTS: The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. CONCLUSION: The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

[Selection pressure analysis of H3N2 influenza virus from China between 1992 and 2012].

OBJECTIVE: In order to investigate the relationship between selection pressure and the prevalence of antigenic clusters, we sequenced and analyzed the H3N2 influenza virus from China between 1992 and 2012. METHODS: The H3N2 influenza virus (n = 1206) in China from 1992 to 2012 was analyzed, include global selection pressure and sites positive selection pressure analysis. RESULTS: Considering all the H3N2 influenza viruses during these 21 years, a total of four amino acid sites subject to positive selection. The global selection pressure varies with the variation of different antigenic clusters and three years with peak bottom selection pressure were identified. CONCLUSION: The global selection pressure rise from the peak bottom, a new antigenic clusters will appear andprevalent in the population, indicating the best time to replace the vaccine strain.

[Reverse genetics platform construction of influenza pandemic virus strain].

OBJECTIVE: Reverse genetics was used to construct the platform of flu pandemic strain A/California/07/2009 (H1N1). METHODS: Eight genes fragements were amplified and ligated with bidirectional vector, recombinant plasmids were co transfected to the 293 T cells and rescued the virus. Gene sequencing, antigenic analysis and growth property were used to evaluate the rescued virus. RESULTS: Rescued virus show the genes sequence correct, keep the same antigenicity and similar growth property compared with wild type virus. CONCLUSION: The pandemic virus reverse genetics platform of A/California/07/2009 (H1N1) were built. Based on this platform, rescued virus hold the similarity of antigenicity and growth ability with wild type virus.